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Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.
- Source :
-
Journal of cellular biochemistry [J Cell Biochem] 1994 May; Vol. 55 (1), pp. 133-45. - Publication Year :
- 1994
-
Abstract
- We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.
- Subjects :
- Animals
Blotting, Western
Cell Line
Centrifugation, Isopycnic
Chemical Fractionation
Chromatography, Gel
Chymotrypsin
Cross Reactions
Cytosol enzymology
Electrophoresis, Polyacrylamide Gel
Killer Cells, Natural enzymology
Lymphocyte Activation
Male
Microscopy, Electron
Proteasome Endopeptidase Complex
Rats
Rats, Inbred F344
Tumor Cells, Cultured
Cysteine Endopeptidases isolation & purification
Interleukin-2 pharmacology
Killer Cells, Natural drug effects
Multienzyme Complexes isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 0730-2312
- Volume :
- 55
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of cellular biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8083294
- Full Text :
- https://doi.org/10.1002/jcb.240550115